Chinese hamster ovary cell lines

ABSTRACT

We provide a Chinese Hamster Ovary (CHO) cell which is capable of higher protein sialylation compared to a wild type Chinese Hamster Ovary cell, such as in the presence of functional GnT 1, in which the CHO cell is obtainable by selection with  Ricinus communis  agglutinin I (RCA-I).

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. §371 National Phase Entry application ofInternational Application No. PCT/SG2009/000348 filed on Sep. 18, 2009,which designates the United States, and which claims the benefit ofpriority under 35 U.S.C. §119(e) of U.S. Provisional Application No.61/098,270 filed on Sep. 19, 2008, and U.S. Provisional Application No.61/183,647 filed on Jun. 3, 2009, the contents of each of which areherein incorporated by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 24, 2014, isnamed 049595-070010-US_SL.txt and is 10,885 bytes in size.

The foregoing applications, and each document cited or referenced ineach of the present and foregoing applications, including during theprosecution of each of the foregoing applications (“application andarticle cited documents”), and any manufacturer's instructions orcatalogues for any products cited or mentioned in each of the foregoingapplications and articles and in any of the application and articlecited documents, are hereby incorporated herein by reference.Furthermore, all documents cited in this text, and all documents citedor reference in documents cited in this text, and any manufacturer'sinstructions or catalogues for any products cited or mentioned in thistext or in any document hereby incorporated into this text, are herebyincorporated herein by reference. Documents incorporated by referenceinto this text or any teachings therein may be used in the practice ofthis invention. Documents incorporated by reference into this text arenot admitted to be prior art.

FIELD

This invention relates to the fields of biotechnology and molecularbiology. The invention in particular relates to Chinese Hamster Ovarycell lines and their use in recombinant protein expression.

BACKGROUND

Proteins are useful in a variety of diagnostic, pharmacologic,agricultural, nutritional, and research applications. Given the highcost of producing proteins, especially therapeutic proteins, even smallincreases in the efficiency of production or in the function andstability of a protein can be valuable.

The function and stability, and hence the utility, of a protein can beaffected by the post-translational addition of sugar residues to theprotein to form a glycoprotein. For example, the addition of terminalsialic acid residues to polysaccharides attached to a glycoproteingenerally increases the protein's lifetime in the bloodstream and can,in particular cases, also affect solubility, thermal stability,resistance to protease attack, antigenicity, and specific activity ofsome glycoproteins. See e.g. Gu and Wang (1998), Biotechnol. and Bioeng.58 (6): 642-48; Morell et al. (1968), J. Biol. Chem. 243 (1): 155-59.

Recombinant glycoprotein proteins and drugs produced by cell lines, suchas Chinese hamster ovary (CHO) cells, generally consist ofdifferentially sialylated isoforms. Poorly sialylated isoforms haveshorter circulatory half-life and are thus less efficacious.

It is therefore desirable to increase the sialic acid content of aglycoprotein, especially a glycoprotein to be used for pharmacologicapplications. Indeed, one of the major research focuses in thebiotechnology field has been how to increase sialylation of recombinantprotein drugs.

SUMMARY

According to a 1^(st) aspect of the present invention, we provide aChinese Hamster Ovary (CHO) cell which is capable of higher proteinsialylation compared to a wild type Chinese Hamster Ovary cell, in whichthe CHO cell is obtainable by selection with Ricinus communis agglutininI (RCA-I). The CHO cell may be capable of higher sialylation in thepresence of functional GnT 1.

The CHO cell may comprise a mutation in the GnT 1 gene.

The mutation in the GnT 1 gene may comprise one or more of the followingmutations at the specified position of a GnT 1 sequence (GenBankaccession number: AF343963): (a) a C to T transition at position 1015;(b) a G to C transversion at position 1300; (c) an A to C transversionat position 638; (d) a C to G transversion at position 784; (e) a T to Atransversion at position 811; (f) an insertion at position 706 resultingin a frame shift from position 236 of the encoded amino acid sequence;(g) a C to T transition at position 1015; (h) a G to A transition atposition 246; (i) a G to A transition at position 258; (j) an A to Ttransversion at position 859.

The GnT 1 gene may comprise a GnT 1 nucleic acid sequence shown in SEQID NO: 1.

The CHO cell may express a GnT 1 protein comprising one or more of thefollowing mutations at the specified position of a GnT 1 sequence(GenBank accession number: AF343963): (a) Ala to Pro at position 434;(b) Asp→Ala at position 213; (c) Arg→Gly at position 262; (d) Trp→Arg atposition 271; (e) frame shift from position 236 resulting from aninsertion at position 706 of an encoding GnT 1 nucleic acid sequence;(f) Gln→STOP at position 339; (g) Trp→STOP at position 82; (h) Trp→STOPat position 86; (i) Lys→STOP at position 287.

The CHO cell may express a GnT 1 protein as shown in SEQ ID NO: 2.

The CHO cell may comprise a nucleic acid sequence encoding a protein ofinterest such as a recombinant protein of interest. The protein ofinterest may comprise erythropoietin (EPO) or interferon-γ (IFN-γ).

The CHO cell may comprise a nucleic acid sequence encoding functionalGnT 1. The nucleic acid sequence encoding the protein of interest andthe nucleic acid sequence encoding functional GnT 1 may be comprised inone expression vector.

The nucleic acid sequence encoding the protein of interest and thenucleic acid sequence encoding functional GnT 1 may be stablytransfected into the CHO cell.

There is provided, according to a 2^(nd) aspect of the presentinvention, a CHO cell line comprising a CHO cell according to the 1^(st)aspect of the invention.

The CHO cell line may comprise a JW 152 cell line (deposited at AmericanType Culture Collection (ATCC) under the Budapest Treaty as accessionnumber PTA-9657 on Dec. 11, 2008), a JW80 cell line, a JW36 cell line, aKFC15002 cell line, a KFC15071 cell line, a KFC5008 cell line, a JW152cell line, a KFC5026 cell line, a KFC20011 cell line or a KFC15047 cellline.

The CHO cell or a CHO cell line may be is adapted to suspension culture.

We provide, according to a 3^(rd) aspect of the present invention, arecombinant protein expressed by a CHO cell or CHO cell line accordingto the 1^(st) or 2^(nd) aspect of the invention.

The recombinant protein may comprise erythropoietin (EPO) orinterferon-γ (IFN-γ).

The recombinant protein may comprise a pKa of 4 or less, 3.5 or less, 3or less, 2.5 or less or 2 or less or a Z-number of greater than 150,such as greater than 160, greater than 170, greater than 180, such asgreater than 190, greater than 200, greater than 210, such as greaterthan 220 or greater than 230, or both.

As a 4^(th) aspect of the present invention, there is provided a methodof expressing a recombinant protein, the method comprising introducing anucleic acid encoding the protein into a CHO cell as set out above,allowing the protein to be expressed from the CHO cell or a descendentthereof, and optionally purifying the protein.

The method may comprise introducing a nucleic acid encoding functionalGnT 1 into the CHO cell.

The method may comprise introducing an expression vector comprising anucleic acid sequence encoding the protein and a nucleic acid sequenceencoding functional GnT 1.

The method may comprise stably transfecting the nucleic acid sequenceencoding the protein of interest and the nucleic acid sequence encodingfunctional GnT 1.

We provide, according to a 5^(th) aspect of the present invention, anucleic acid comprising a sequence shown in SEQ ID NO: 1, or a sequencecomprising a GnT 1 sequence together with a mutation set out in Column 2of Table D1, or a variant, homologue, derivative or fragment thereof.

The present invention, in a 6^(th) aspect, provides a polypeptidecomprising a sequence as shown in SEQ ID NO: 2, or a sequence comprisinga GnT 1 sequence together with a mutation set out in Column 3 of TableD1, or a variant, homologue, derivative or fragment thereof.

In a 7^(th) aspect of the present invention, there is provided a methodof providing a CHO cell or cell line, the method comprising culturingCHO cells in the presence of Ricinus communis agglutinin I (RCA-I) andselecting cells which survive the culture.

The method may comprise culturing CHO cells in the presence of Ricinuscommunis agglutinin I (RCA-I) and selecting cells which survive theculture.

The method may comprise exposing the CHO cells to RCA-I at aconcentration of between 0.1 μg/ml to 100 μg/ml, for example up to 50μg/ml or up to 20 μg/ml, such as 10 μg/ml or 5 μg/ml.

The method may comprise exposing the CHO cells to RCA-I for a period offrom an hour, a few hours (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12),overnight, to a few days, such as 2 days or 3 days, such as overnight.

The method may comprise further comprising selecting cells which do notreact with RCA-I in an agglutination test.

According to an 8^(th) aspect of the present invention, we provide a CHOcell or CHO cell line obtainable by a method set out above.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA and immunology, which are within thecapabilities of a person of ordinary skill in the art. Such techniquesare explained in the literature. See, for example, J. Sambrook, E. F.Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual,Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel,F. M. et al. (1995 and periodic supplements; Current Protocols inMolecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York,N.Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation andSequencing: Essential Techniques, John Wiley & Sons; J. M. Polak andJames O'D. McGee, 1990, In Situ Hybridization: Principles and Practice;Oxford University Press; M. J. Gait (Editor), 1984, OligonucleotideSynthesis: A Practical Approach, Irl Press; D. M. J. Lilley and J. E.Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesisand Physical Analysis of DNA Methods in Enzymology, Academic Press;Using Antibodies: A Laboratory Manual: Portable Protocol NO. I by EdwardHarlow, David Lane, Ed Harlow (1999, Cold Spring Harbor LaboratoryPress, ISBN 0-87969-544-7); Antibodies: A Laboratory Manual by Ed Harlow(Editor), David Lane (Editor) (1988, Cold Spring Harbor LaboratoryPress, ISBN 0-87969-314-2), 1855. Handbook of Drug Screening, edited byRamakrishna Seethala, Prabhavathi B. Fernandes (2001, New York, N.Y.,Marcel Dekker, ISBN 0-8247-0562-9); and Lab Ref: A Handbook of Recipes,Reagents, and Other Reference Tools for Use at the Bench, Edited JaneRoskams and Linda Rodgers, 2002, Cold Spring Harbor Laboratory, ISBN0-87969-630-3. Each of these general texts is herein incorporated byreference.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a photograph showing that transiently expressed erythropoietin(EPO) in JW152 cell line is sensitive to endoglucosidase H (Endo H)treatment. Western blot of transiently expressed EPO: from CHO K-1 cells(Lane 1), from JW152 cells (Lane 2), Endoglucosidase H treated EPO fromCHO-K1 (Lane 3), Endoglucosidase H treated EPO from JW152 (Lane 4).JW152 expressed EPO is sensitive to Endo H treatment suggesting that theglycan structure found on the EPO molecule is of the high mannose type.It is hence suspected that gene(s) from the early part of theglycosylation pathway is defective.

FIG. 2 is a photograph showing that JW152 cells lack a functional GnT Igene. Recombinant EPO is analyzed by the IEF/Western blot assay. Thelane on the far left (CHO WT) shows the EPO expressed by the wild typeCHO cells as a control. The second lane shows the EPO produced by theJW152 cells. Incomplete glycosylation in JW152 cells is obvious bycomparing the two lanes. For the complementation test, each of severalglycosylation related genes (as indicated) is co-transfected with theEPO construct into JW152 cells. Only GnT I is able to complement thegenetic defect in JW152 cells, suggesting these cells lack a functionalGnT I gene. Another very important observation from this IEF results isthat in the presence of GnT I, JW152 cells sialylate EPO much betterthan the wild type cells.

FIG. 3 is a photograph showing that improvement in sialylation afterco-transfecting with GnT I is observed for JW152 but not for CHO-K1. Thesialylation pattern of EPO produced in CHO-K1, CHO-K1 with co-expressionof GnT I. EPO produced in JW152 cells and in JW152 cells with theco-expression of GnT I. The EPO sialylation patterns of CHO-K1 with andwithout co-expression of GnT I appear to be the same and so, theoverexpression of GnT I is not responsible for the betterment insialylation seen in lane 4. Results suggest that in the presence of GnTI, JW152 cells sialylate recombinant proteins much better than the wildtype cells. Treating samples with neuraminidase, which cleaves offsialic acid, results in EPO bands in the acidic region being reduced tothe basic region after cleaving off sialic acid, showing that the highersialylated forms of EPO are indeed focused in the acidic region of thegel.

FIG. 4 is a photograph showing that EPO expression in the presence offunctional GnT I of previously published Lec 1 mutant which has a GnT Idefect is shown to be also highly sialylated. Transient EPO expressed inCHO-K1, Lec 1 and Lec 1 with the restoration of functional GnT I. Lec 1is previously reported and had been independently isolated using adifferent lectin by another group headed by Pamela Stanley.

FIG. 5 is a photograph showing that all nine CHO glycosylation mutants,resistant to RCA and bearing distinct mutations in GnT I gene, lead toincomplete sialylation. Transiently expressed EPO in JW152, JW36, JW80,KFC5008, KFC5026, KFC15002, KFC15047, KFC15071, KFC 20011, CHO-K1. EPOproduced in nine CHO glycosylation mutants selected with RCA lectin,each bearing a different mutation in the GnT I gene lead to a loss inGnT I function. Incomplete sialylation is observed in these cell linesas compared to CHO wild-type (extreme right). EPO clinical standard ison extreme left. RCA lectin yields mainly GnT I deficient mutants.

FIG. 6 is a photograph showing that all nine CHO glycosylation mutants,after restoring GnT I function transiently express EPO that is bettersialylated than that in CHO-K1.

FIG. 7 is a photograph showing that JW152, with restored GnT I function,also sialylates EPO-Fc fusion protein better than CHO-K1. EPO-Fc fusionprotein produced in CHO wild-type compared alongside with that producedin JW152 before and after rescue. The results demonstrate that thesuperior sialylation by rescued JW152 is maintained even with adifferent model glycoprotein.

FIG. 8 is a photograph showing that HPAEC chromatogram showing bettersialylated glycans cleaved from EPO-Fc expressed in JW152 cellsco-expressing functional GnT I. EPO-Fc transiently expressed in CHO-K1and JW152, contransfected with GnT I, is purified using affinitypurification via a protein A-bound chromatography column. Glycans on theequal amounts of EPO-Fc are cleaved by treatment with Peptide:N-Glycosidase F (PNGase F), and separated, using High pH Anion ExchangeChromatography (HPAEC), according to their number of sialic acidsattached to the glycans. The chromatogram shows distinctly higher peaksfor the JW152 sample in the 4S group and lower peaks in the 1S groupwhen comparing with the CHO-K1 sample. Arrows point to an internalcontrol, raffinose. Samples are labeled as shown.

FIG. 9 is a photograph showing recombinant EPO expressed by 10 randomlypicked clones isolated by RCA-I. Top gel, EPO expressed in 10 differentmutant lines. Cells are transfected with a construct that expresses EPOalone. Bottom gel, EPO expressed in the same set of cell lines that aretransfected with pEIG. This construct expresses both EPO and GnT I.

FIG. 10 is a photograph showing that EPO produced in 10 different JW152clones that are stably transfected with a construct that expresses bothEPO and GnT I. JW152 cells are transfected with a construct called pEIG.In pEIG, a transcription unit containing EPO-IRES-GnT I (EIG) is clonedinto pcDNA3.1 downstream of the CMV promoter. After transfection, stableclones are selected and picked. The sialylation patterns of EPO producedby 10 such randomly picked clones are analyzed by the IEF/Western blotassay. The results show that all the EPO samples produced by differentstable clones are highly sialylated. This means that better sialylationis maintained at stable expression of EPO in the presence of functionalGnT I.

FIG. 11 is a photograph showing that stable expression of EPO in CHO-K1shows a sialylation profile that is inferior to that of JW152 withrestored GnT I function. CHO-K1 cells are transfected with expressionvector containing EPO coding sequence and put under selection. Clonesare isolated and EPO from nine randomly selected stable clones isanalysed with IEF. The results show that all the EPO samples produced bydifferent stable clones vary in overall sialylation but in general arenot as well sialylated as those in FIG. 10.

FIG. 12 is a photograph showing that EPO produced in four JW152 stablecell lines that have been adapted to suspension batch culture in proteinfree medium. The sialylation pattern observed for stable JW152 celllines in attached culture is maintained in suspension culture.

SEQUENCE LISTING

SEQ ID NO: 1 is a nucleic acid sequence of aN-acetylglucoaminyltransferase I cDNA from CHO JW152 cells.

SEQ ID NO: 2 is an amino acid sequence of N-acetylglucoaminyltransferaseI encoded by SEQ ID NO: 1.

DETAILED DESCRIPTION

Using a cytotoxic lectin, RCA-I, we have isolated a novel CHO mutantcell line, JW152, from CHO-K1 cells.

Recombinant EPO produced by JW152 cells that are stably transfected withEPO and GnT I cDNAs contains the highly sialylated isoforms. Several ofthese stably transfected lines have been adapted in suspension cultureand grown in serum-free medium. The EPO produced by these cells areanalyzed by an IEF assay. The results showed that EPO produced inserum-free medium remained highly sialylated.

These results suggest that JW152 cells have the potential to become ahost cell line for producing proteins, such as highly sialylatedproteins including glycoprotein drugs.

The cell line JW152 was deposited on 11 Dec. 2008 at the American TypeCulture Collection, 10801 University Boulevard, Manassas, Va.20110-2209, United States of America under the accession number PTA-9657as the International Deposition Number under the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure.

We therefore provide for a Chinese Hamster Ovary (CHO) cell or cell linewhich is capable of higher protein sialylation compared to a wild typeChinese Hamster Ovary cell.

The CHO cell capable of higher sialylation may be produced by a suitableselection method such as a RCA-1 selection method. Such a method isdescribed in further detail in “CHO Cells and Cell Lines” below.Therefore, RCA-I can be used to isolate CHO glycosylation mutant cellsproduce highly sialylated recombinant proteins in the presence of GnT I.

Such a selection method is therefore specifically included in themethods and compositions described here.

We therefore provide for a Chinese Hamster Ovary (CHO) cell or cell linewhich is capable of higher protein sialylation compared to a wild typeChinese Hamster Ovary cell, the CHO cell being obtainable by selectionwith Ricinus communis agglutinin I (RCA-I). In general, we provide aRCA-I resistant CHO cell or cell line, such as an RCA-I resistant CHO-K1cell or cell line.

Genetic analysis has revealed a dysfunctionalN-Acetyl-glucosaminyltransferase I (GnT I) gene in JW152 cells.Molecular cloning of the GnT I cDNA from the mutant cells identified apoint mutation that results in a premature stop codon. As a result, theJW152 cells can only synthesize a truncated version of GnT I proteinwith only 338 amino acids, rather than the normal protein which contains447 amino acids.

Using RCA-I we have isolated many more CHO mutant lines (about 100clones). Genetic analyses showed that they all lack functional GnT Igene. Many of them carry a different point mutation in the coding regionof GnT I gene, suggesting that they derived from different originalclones. Yet, they all dramatically improved sialylation of recombinantproteins in the presence of GnT I. The mutations in the GnT 1 genes fromthese further CHO cell lines are shown in Table D1 below.

Accordingly, we provide for a Chinese Hamster Ovary (CHO) cell which iscapable of higher protein sialylation in the presence of functional GnT1 compared to a wild type Chinese Hamster Ovary cell, the CHO cellcomprising a mutation in the GnT 1 gene. We further provide for aChinese Hamster Ovary (CHO) cell or cell line which is capable of higherprotein sialylation compared to a wild type Chinese Hamster Ovary cell,the CHO cell being obtainable by selection with Ricinus communisagglutinin I (RCA-I) and comprising a mutation in the GnT 1 gene.

The mutation in the GnT 1 gene may comprise any point mutation,deletion, inversion, etc. The mutation in the GnT 1 gene may encode apartially functioning, or non-functioning GnT 1 polypeptide. Themutation in the GnT 1 gene may encode a truncated GnT 1 polypeptide.

We provide for CHO cells and cell lines comprising each of these mutantCHO cell lines and clones. We provide for specific cell lines derivedfrom RCA-I selection and capable of higher sialylation compared to wildtype or native CHO cells or parental cells. We provide for mutant CHOnucleic acid and polypeptide sequences comprised in such cells or celllines, as described in further detail below.

The CHO cell or cell line may comprise a JW152 cell line. It maycomprise a JW80 cell line. It may comprise a JW36 cell line. It maycomprise a KFC15002 cell line. It may comprise a KFC15071 cell line. Itmay comprise a KFC5008 cell line. It may comprise a JW152 cell line. Itmay comprise a KFC5026 cell line. It may comprise a KFC20011 cell line.It may comprise a KFC15047 cell line.

The CHO cell or cell line may be transfected with a nucleic acidencoding a protein of interest, for example a heterologous orrecombinant protein. Such a protein might comprise a glycoprotein. TheCHO cell or cell line may be transfected or co-transfected with anucleic acid encoding a functional or full length or wild type GnT 1sequence.

As noted above, we provide for the nucleic acid themselves, e.g., anucleic acid encoding a mutant GnT 1 gene, or a fragment, variant,derivative or homologue of such a nucleic acid. The nucleic acidencoding a mutant GnT 1 gene, fragment, variant, derivative or homologuemay cause a CHO cell comprising it to be capable of higher sialylation,compared to a CHO cell which does not comprise such a nucleic acid, forexample a wild type CHO cell. The nucleic acid encoding the mutant GnT 1gene may comprise a sequence shown as SEQ ID NO: 1 or a variant,homologue, derivative or fragment thereof.

SEQ ID NO: 1

The coding region of N-acetylglucoaminyltransferase I (Mgat1, GenBank:AF343963) mRNA isolated from the CHO JW152 cells. In these mutant cells,a C to T point mutation at position 1015 was identified (shown in bold):

ATGCTGAAGAAGCAGTCTGCAGGGCTTGTGCTTTGGGGTGCTATCCTCTTTGTGGGCTGGAATGCCCTGCTGCTCCTCTTCTTCTGGACACGCCCAGCCCCTGGCAGGCCCCCCTCAGATAGTGCTATCGATGATGACCCTGCCAGCCTCACCCGTGAGGTGTTCCGCCTGGCTGAGGACGCTGAGGTGGAGTTGGAGCGGCAGCGGGGGCTGTTGCAGCAAATCAGGGAGCATCATGCTTTGTGGAGACAGAGGTGGAAAGTGCCCACCGTGGCCCCTCCAGCCTGGCCCCGTGTGCCTGCGACCCCCTCACCAGCCGTGATCCCCATCCTGGTCATTGCCTGTGACCGCAGCACTGTCCGGCGCTGCTTGGATAAGTTGTTGCACTATCGGCCCTCAGCTGAGCATTTCCCCATCATTGTCAGCCAGGACTGCGGGCACGAAGAGACAGCACAGGTCATTGCTTCCTATGGCAGTGCAGTCACACACATCCGGCAGCCAGACCTGAGTAACATCGCTGTGCCCCCAGACCACCGCAAGTTCCAGGGTTACTACAAGATCGCCAGGCACTACCGCTGGGCACTGGGCCAGATCTTCAACAAGTTCAAGTTCCCAGCAGCTGTGGTAGTGGAGGACGATCTGGAGGTGGCACCAGACTTCTTTGAGTACTTCCAGGCCACCTACCCACTGCTGAGAACAGACCCCTCCCTTTGGTGTGTGTCTGCTTGGAATGACAATGGCAAGGAGCAGATGGTAGACTCAAGCAAACCTGAGCTGCTCTATCGAACAGACTTTTTTCCTGGCCTTGGCTGGCTGCTGATGGCTGAGCTGTGGACAGAGCTGGAGCCCAAGTGGCCCAAGGCCTTCTGGGATGACTGGATGCGCAGACCTGAGCAGCGGAAGGGGCGGGCCTGTATTCGTCCAGAAATTTCAAGAACGATGACCTTTGGCCGTAAGGGTGTGAGCCATGGGCAGTTCTTTGATCAGCATCTTAAGTTCATCAAGCTGAACCAGTAGTTCGTGTCTTTCACCCAGTTGGATTTGTCATACTTGCAGCGGGAGGCTTATGACCGGGATTTCCTTGCCCGTGTCTATAGTGCCCCCCTGCTACAGGTGGAGAAAGTGAGGACCAATGATCAGAAGGAGCTGGGGGAGGTGCGGGTACAGTACACTAGCAGAGACAGCTTCAAGGCCTTTGCTAAGGCCCTGGGTGTCATGGATGACCTCAAGTCTGGTGTCCCCAGAGCTGGCTACCGGGGCGTTGTCACTTTCCAGTTCAGGGGTCGACGTGTCCACCTGGCACCCCCACAAACCTGGGAAGGC TATGATCCTAGCTGGAATTAG

We further provide for mutant GnT 1 polypeptides, as well as fragments,variants, derivatives and homologoues thereof. The mutant GnT 1polypeptide, fragment, variant, derivative or homologue may cause a CHOcell comprising it to be capable of higher sialylation, compared to aCHO cell which does not comprise such a polypeptide, for example a wildtype CHO cell. The mutant GnT 1 polypeptide may comprise a sequenceshown as SEQ ID NO: 2 or a variant, homologue, derivative or fragmentthereof.

SEQ ID NO: 2

The N-acetylglucoaminyltransferase I (GnT I) protein encoded by themutated gene in CHO JW152 cells. As a result of the point mutation(C1015T), JW152 cells only produce a truncated version of GnT I whichcontains only 338 amino acids rather than the normal protein thatcontains 447 amino acids. The C-terminal portion in bold is nottranslated in JW152 cells.

MLKKQSAGLVLWGAILFVGWNALLLLFFWTRPAPGRPPSDSAIDDDPASLTREVFRLAEDAEVELERQRGLLQQIREHHALWRQRWKVPTVAPPAWPRVPATPSPAVIPILVIACDRSTVRRCLDKLLHYRPSAEHFPIIVSQDCGHEETAQVIASYGSAVTHIRQPDLSNIAVPPDHRKFQGYYKIARHYRWALGQIFNKFKFPAAVVVEDDLEVAPDFFEYFQATYPLLRTDPSLWCVSAWNDNGKEQMVDSSKPELLYRTDFFPGLGWLLMAELWTELEPKWPKAFWDDWMRRPEQRKGRACIRPEISRTMTFGRKGVSHGQFFDQHLKFIKLNQQFVSFTQLDLSYLQREAYDRDFLARVYSAPLLQVEKVRTNDQKELGEVRVQYTSRDSFKAFAKALGVMDDLKSGVPRAGYRGVVTFQFRGRRVHLAPPQTWEG YDPSWN

Several of the JW152-pEIG stable lines shown in FIG. 2 have been adaptedin suspension culture and grown in serum-free medium. The EPO producedin serum-free medium remained highly sialylated. We therefore providefor mutant CHO cells and cell lines derived from RCA-I selection, whichhave been adapted to suspension culture, or growth in semi-solid medium.

In conclusion, we have developed a novel method to isolate glycosylationmutant cells from CHO cells. All CHO cells that survive RCA-I treatmenthave very similar characteristics. First, they all lack a functional GnTI gene. Second, they all sialylate their recombinant proteins betterthan the wild type CHO cells in the presence of GnT I. This featureremains the same both in transient transfection and in stablytransfected cells.

The CHO glycosylation mutant cells isolated with RCA-I, such as JW152cells, can produce recombinant glycoproteins with high degree ofsialylation as long as GnT I is present. This method can be used toproduce recombinant glycoproteins in which sialic acid content isimportant for the efficacy. These proteins include EPO, IEF-γ, FactorVIII etc.

CHO Cells and Cell Lines

The CHO cells and cell lines described here may be made by any suitablemeans. For example, the CHO cells and cell lines may be produced byselection using a suitable agglutinating agent, such as agglutinin I.The agglutinin may comprise any suitable agglutinin, such as Ricinuscommunis agglutinin I (RCA-I).

We therefore provide a method of providing a CHO cell or cell line, themethod comprising culturing CHO cells in the presence of Ricinuscommunis agglutinin I (RCA-I) and selecting cells which survive theculture.

The CHO cells and cell lines described here may be made by treating astarting or parent cell with Ricinus communis agglutinin I (RCA-I) andselecting cells that survive such treatment. Such surviving cells may befurther cloned and made into cell lines. The selected cells and celllines may comprise higher sialylation activity as described in thisdocument. The selected cells and cell lines may comprise mutant GnT 1genes and polypeptides, as described in this document.

For example, the CHO cells or cell lines described here may be selectedby exposing a parent cell line to Ricinus communis agglutinin I (RCA-I)at a suitable concentration for a suitable period.

The RCA-1 concentration could range from between 0.1 μg/ml to 100 μg/ml,for example up to 50 μg/ml or up to 20 μg/ml. Examples of specificconcentrations include 10 μg/ml and 5 μg/ml.

The period of incubation or exposure to RCA-1 could be from an hour, afew hours (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12), overnight, to afew days, such as 2 days or 3 days.

In general, the period and concentration can be adjusted to eliminatethe majority of CHO cells, but to enable a small proportion of cells,which are resistant to RCA-I, to survive and form colonies. Within this,the concentration and period of exposure and selection may be varied,but generally, the higher the concentration of RCA-I, the lower theperiod of exposure is necessary, and vice versa.

The selection could be done on any suitable starting cell or cell line,but this will generally be a CHO cell or cell line. Any known CHO cellor cell line could be used as a starting point or parent cell, includingCHO-K1. Other suitable starting cells could include, but are not limitedto the following (ECACC accession numbers in brackets): CHO (85050302),CHO (PROTEIN FREE) (00102307), CHO-K1 (85051005), CHO-K1/SF (93061607),CHO/dhFr-(94060607), CHO/dhFr-AC-free (05011002), RR-CHOKI (92052129).

Following selection, the surviving cells are allowed to grow and formcolonies following which they may be picked. The time allowed for thiswill vary, but will generally be long enough for colonies to grow to apickable size. Examples of such times are 5 days, 7 days, 9 days, 11days, 13 days, one week, two weeks, three weeks or more.

The picking may be done manually, or it may be automated through use ofrobots, such as CLONEPIX (Genetix, New Milton, Hampshire, UK). Thepicked colonies may be further cloned, further screened, characterisedand cultured, etc.

The selected cells may be subjected to further tests. For example, theymay be subjected to agglutination tests using RCA-I to confirm themutant cells no longer react with RCA-I.

As a specific example, which is not intended to be limiting, CHO-K1cells may be cultured, for example in 6-well plates, to confluence.Culture media may be changed to serum-free DMEM. Ricinus communisagglutinin I (RCA-I, EY Laboratories) may be added into the media toreach a final concentration of 10 μg/gml. This may be incubated withcells overnight. The serum-free DMEM containing RCA-I may be replaced,for example with fresh DMEM with 10% FBS. Nine days later, colonies ofthe CHO cells that survive the RCA-I treatment may be picked andcultured, for example in 24 well plates.

These cells may be subjected to further tests, such as agglutinationtests using RCA-I to confirm the mutant cells no longer react withRCA-I. We therefore provide for a method of providing a CHO cell or cellline, the method comprising culturing CHO cells in the presence ofRicinus communis agglutinin I (RCA-I), selecting cells which survive theculture and which do not react with RCA-I in an agglutination test.

The RCA-I selected CHO cells and CHO cell lines may be tested for theirsialylation behaviour, by for example expressing a protein of interestand determining the degree of sialylation. This may be done by themethods described in “Sialylation” below.

We therefore provide for a method of providing a CHO cell or cell line,the method comprising culturing CHO cells in the presence of Ricinuscommunis agglutinin I (RCA-I), selecting cells which survive the cultureand selecting those cells or cell lines which display high sialylationbehaviour, for example high Z-number or low pI of expressed proteins.

The GnT 1 gene in such selected cells may be cloned and sequenced, usingmethods known in the art. The GnT 1 gene may comprise a mutant GnT 1gene as described here.

We therefore provide for a method of providing a CHO cell or cell line,the method comprising culturing CHO cells in the presence of Ricinuscommunis agglutinin I (RCA-I), selecting cells which survive the cultureand selecting those cells or cell lines which comprise mutant GnT 1genes as described herein.

Mutant CHO Cells and Cell Lines

We provide for a CHO cell or cell line derived from RCA-I selection, asdescribed above. Such a cell line could include a JW152 cell line, orany of the cell lines set out in Table D1 below, including a JW80 cellline, a JW36 cell line, a KFC15002 cell line, a KFC15071 cell line, aKFC5008 cell line, a JW152 cell line, a KFC5026 cell line, a KFC20011cell line or a KFC15047 cell line.

Protein Expression

The CHO cells described here may be used as host cells for expression ofany protein of interest. This may be done by means known in the art.

Protein expression in CHO cells and cell lines is well described in theliterature, and the skilled person will have little difficulty in usingthe CHO cells and cell lines described here as hosts for proteinexpression. Thus, for example, the CHO cells and cell lines may betransfected by means known in the art with expression vectors capable ofexpressing the protein of interest.

The CHO cells and cell lines may further be capable of expressing wildtype or functional GnT 1, for example, the sequence set out in GenBankaccession number AF343963. This may be done by transfecting the CHOcells and cell lines with an expression vector encoding GnT 1. This maybe on the same or different vector as that which contains the nucleicacid encoding the protein of interest

Any suitable protein may be expressed using the CHO cells described hereas host cells. The protein may comprise a heterologous protein. Theprotein may comprise a recombinant protein. The protein may comprise anengineered protein. The protein may comprise a glycoprotein.

Examples include heterologous proteins of therapeutic or pharmacologicalinterest. Proteins which may be expressed include anti-EGFR mAb,α-glucosidase, laronidase, Ig-CTLA4 fusion,N-acetylgalactosamine-4-sulfatase, luteinizing hormone, anti-VEGF mAb,Factor VIII, anti-lgE mAb, anti-CD11a mAb, α-galactosidase,interferon-β, anti-TNFα mAb, erythropoietin, anti-CD52 mAb, Factor VIII,tissue plasminogen activator, anti-HER2 mAb, TNFα receptor fusion,Factor IX, follicle stimulating hormone, anti-CD20 mAb, interferon-β,β-glucocerebrosidase, deoxyribonuclease I, etc.

For example, we describe the expression of erythropoietin (EPO),interferon-γ and Factor VIII with CHO cells and cell lines describedhere. We also describe highly sialylated forms or isoforms oferythropoietin (EPO), interferon-γ and Factor VIII expressed from CHOcells and cell lines described here.

Sialic Acid

The term “sialic acid” is intended to refer to any member of a family ofnine-carbon carboxylated sugars.

The most common member of the sialic acid family is N-acetylneuraminicacid (2-keto-5-acetamido-3,5-dideoxy-D-glycero-D-galactononulopyranos-1-onic acid, often abbreviatedas Neu5Ac, NeuAc, or NANA). A second member of the family isN-glycolylneuraminic acid (Neu5Gc or NeuGc), in which the N-acetyl groupof NeuAc is hydroxylated. A third sciatic acid family member is2-keto-3-deoxy-nonulosonic acid (KDN). 17

Also included are 9-substituted sialic acids such as a9-O—C1-C6-acyl-Neu5Ac like 9-O-lactyl-Neu5Ac or 9-O-acetyl-Neu5Ac,9-deoxy-9-fluoro-Neu5Ac and 9-azido-9-deoxy-Neu5Ac. For review of thesialic acid family, see, e.g., Varki; Glycobiology 2 1992; 25-40; SialicAcids: Chemistry, Metabolism and Function, R. Schauer, Ed.

Sialylation

The degree of sialylation of a protein may be measured by various means,such as using Z-numbers or expressing the isoelectric point of theprotein.

We therefore provide for protein expression using the CHO cells and celllines described here, such protein expression comprising highlysialylated forms or isoforms of proteins of interest. Protein expressionfrom CHO cells and CHO cell lines described here enables production ofproteins such as glycoproteins, for example erythropoietin (EPO),interferon-γ and Factor VIII, which have high Z-numbers or low pIs, orboth.

Z-number

The parameter Z-number provides a measure of how many of the antennae ofthe carbohydrate moieties in a glycoprotein bear charged residues, suchas sialic acid.

To determine Z-number, the carbohydrate moieties are released from thepeptide, as above, and labelled, if desired. The mixture is thenseparated by ion exchange chromatography, allowing the separation ofspecies on the basis of charge. Visualisation of the eluted peaks may beby virtue of a label, as mentioned above, or may be by some othermethod, such as mass-spectrometry.

A chromatogram is then analysed by integrating the peaks associated withmono- di- tri- and tetra-charged carbohydrate species. The percentage ofthe total carbohydrate represented by each species can then be used tocalculate Z-number according to the following equation: Z=P′mono+2P′di+3 P′tri+4 P′tetra wherein Z is Z-number, and P′mono, P′di, P′triand P′tetra are the percentage of total carbohydrate that is mono-, di-,tri- and tetra-charged respectively.

A high Z-number indicates that a large number of antennae bear chargedresidues, and that the glycoprotein will therefore be highly charged,and in the case of sialic acid residues, acidic.

The CHO cells described here are capable of expressing proteins with ahigh degree of sialylation. Thus, for example, the CHO cells may expressproteins with high Z-number values such as greater than 150, such asgreater than 160, greater than 170, greater than 180, such as greaterthan 190, greater than 200, greater than 210, such as greater than 220,greater than 230, etc.

Isoelectric Point

The isoelectric point (pI) of a protein may also be used as a measure ofits sialylation. The higher the degree of sialylation, the more acidicit is and the lower its pI.

Proteins expressed by the CHO cells described here have significantlylower pi profiles than their normal counterparts, e.g., native proteins,or counterpart proteins expressed by wild type CHO cells. For example,the proteins expressed by the CHO cells and cell lines described heremay have low pIs, such as pIs of 4.5 or less, such as 4.3 or less, 4.1or less, 4.0 or less, 3.8 or less, 3.6 or less, 3.4 or less, 3.2 orless, 3.0 or less, 2.8 or less, 2.6 or less, etc. The pIs may be ofindividual protein molecules, or a batch or fraction of them, or anaverage pI of an expressed lot of protein.

The expressed proteins may be isolated from a mixture of isoforms usinga number of methods that will be known to one skilled in the art. Forexample, isoelectric focussing, chromatofocussing or ion-exchangechromatography may be used to separate the isoforms on the basis of pl.The different fractions can be analysed for sialic acid content, and thedesired fractions selected for use.

Mutant GnT 1 Sequences

We disclose mutant GnT 1 sequences comprising mutant GnT 1 amino acidsequences and mutant GnT1 nucleic acid sequences.

Example mutant GnT 1 amino acid sequences include the sequence shown asSEQ ID NO: 2, as well as the sequences comprising the mutations shown incolumn 3 of Table D1 below.

Example mutant GnT 1 nucleic acid sequences include the sequence shownas SEQ ID NO: 1, as well as the sequences comprising the mutations shownin column 2 of Table D1 below.

Table D1 shows the mutations in the GnT 1 sequence of clones isolatedfrom selection with RCA-I, as described in the Examples below.

Corresponding mutations are tabulated alongside showing respectivenucleotide and amino acid mutation and possible location of thedisruption in secondary structure/interaction or the resulting loss inamino acids in the case of a stop codon mutation.

A minimum of 4 bacteria colonies were sequenced to ensure that themutations found were not due to PCR error.

TABLE D1 Table of mutations found in GnT1 mutants from clones derivedfrom RCA-I screening (Example 10 below). Nine CHO glycosylation mutantswith different mutations in the GnT1 gene. Point mutations leading toloss in function were found in four cell lines (JW80, JW36, KFC15002,KFC15071). A point insertion resulting in the generation of a stop codonwas found in KFC 5008. Mutations leading to a premature stop codon werealso found in another four cell lines. (JW152, KFC5026, KFC20011,KFC15047). Mutant CHO Cell Polypeptide Mutation Line DNA Mutation(position, mutation) Comment JW80 G1300C Position 434 Domain 2 Ala→Proβ14 JW36 A638C Position 213 Disruption of DxD Motif, Asp→Ala similar toJW98, JW191 KFC15002 C784G Position 262 Domain 1 β6, Arg→Gly similar toKFC15008 (aka 15008), KFC7501 (aka 7501) and KFC12008 KFC15071 T811APosition 271 Domain 1 Trp→Arg β7 KFC5008 _706C Insertion at 706 bp Stopcodon generated at 245 Frame shift from 236 aa a.a. Asp→STOP JW152C1015T Position 339 108 a.a. missing from C Gln→STOP terminal KFC5026G246A Position 82 365 a.a. missing from C Trp→STOP terminal KFC20011G258A Position 86 361 a.a. missing from C Trp→STOP terminal KFC15047A859T Position 287 160 a.a. missing, similar to Lys→STOP KFC15026 andKFC15072Mutant GnT 1 Polypeptides

The CHO cells and cell lines comprise mutant GnT 1 polypeptides.

We therefore provide generally for a mutant GnT 1 polypeptide, togetherwith fragments, homologues, variants and derivatives thereof. Thesepolypeptide sequences may comprise the polypeptide sequences disclosedhere, and particularly in the sequence listings.

The mutant GnT 1 polypeptide may comprise one or more changes comparedto the wild type GnT 1 sequence. Such mutations may result from stopcodons being introduced in the encoding nucleic acid sequence andconsequent premature termination of translation of the GnT 1 mRNA.

The mutant GnT 1 polypeptide may be shorter than a wild type GnT 1polypeptide. It may be a truncated version of wild type GnT 1polypeptide. The length of the mutant GnT 1 polypeptide may be 90% orless, 80% or less, 70% or less, etc than the wild type sequence.

For example, a mutant GnT 1 polypeptide may be missing 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or moreC-terminal residues compared to full length or wild type GnT 1polypeptide.

The mutant GnT 1 polypeptide may for example comprise a sequence set outin SEQ ID NO: 2. This is the mutant GnT 1 polypeptide sequence from thecell line JW152. The mutant GnT 1 polypeptide may comprise a GnT 1sequence comprising a mutation set out at column 3 of Table D1 above.

It will be understood that the mutant GnT 1 polypeptide sequencesdisclosed here are not limited to the particular sequences set forth inthe sequence listing, or fragments thereof, or sequences obtained frommutant GnT 1 protein, but also include homologous sequences obtainedfrom any source, for example related cellular homologues, homologuesfrom other species and variants or derivatives thereof, provided thatthey have at least one of the biological activities of mutant GnT 1, asthe case may be.

This disclosure therefore encompasses variants, homologues orderivatives of the amino acid sequences set forth in the sequencelistings, as well as variants, homologues or derivatives of the aminoacid sequences encoded by the nucleotide sequences disclosed here. Sucha sequences is generally referred to as a “mutant GnT 1 sequence”.

The length of the mutant GnT 1 polypeptide may be 90% or less, 80% orless, 70% or less, etc than a corresponding wild type sequence.

For example, a mutant GnT 1 nucleic acid may encode a mutant GnT 1polypeptide that is missing 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, 100 or more C-terminal residues.

Biological Activities

In some embodiments, the sequences comprise at least one biologicalactivity of mutant GnT 1, as the case may be. The biological activitymay comprise improved ability to express proteins with highersialylation compared to wild type GnT 1

Homologues

The polypeptides disclosed include homologous sequences obtained fromany source, for example related viral/bacterial proteins, cellularhomologues and synthetic peptides, as well as variants or derivativesthereof.

In the context of the present document, a homologous sequence orhomologue is taken to include an amino acid sequence which is at least60, 70, 80 or 90% identical, such as at least 95 or 98% identical at theamino acid level over at least 30, such as 50, 70, 90 or 100 amino acidswith GnT 1, as the case may be, for example as shown in the sequencelisting herein. In the context of this document, a homologous sequenceis taken to include an amino acid sequence which is at least 15, 20, 25,30, 40, 50, 60, 70, 80 or 90% identical, such as at least 95 or 98%identical at the amino acid level, such as over at least 15, 25, 35, 50or 100, such as 200, 300, 400 or 500 amino acids with the sequence ofGnT 1.

Although homology can also be considered in terms of similarity (i.e.amino acid residues having similar chemical properties/functions), inthe context of the present document it is possible to express homologyin terms of sequence identity. In some embodiments, the sequenceidentity is determined relative to the entirety of the length therelevant sequence, i.e., over the entire length or full length sequenceof the relevant gene, for example.

Homology comparisons can be conducted by eye, or more usually, with theaid of readily available sequence comparison programs. Thesecommercially available computer programs can calculate % homologybetween two or more sequences.

% homology may be calculated over contiguous sequences, i.e. onesequence is aligned with the other sequence and each amino acid in onesequence directly compared with the corresponding amino acid in theother sequence, one residue at a time. This is called an “ungapped”alignment. Typically, such ungapped alignments are performed only over arelatively short number of residues (for example less than 50 contiguousamino acids).

Although this is a very simple and consistent method, it fails to takeinto consideration that, for example, in an otherwise identical pair ofsequences, one insertion or deletion will cause the following amino acidresidues to be put out of alignment, thus potentially resulting in alarge reduction in % homology when a global alignment is performed.Consequently, most sequence comparison methods are designed to produceoptimal alignments that take into consideration possible insertions anddeletions without penalising unduly the overall homology score. This isachieved by inserting “gaps” in the sequence alignment to try tomaximise local homology.

However, these more complex methods assign “gap penalties” to each gapthat occurs in the alignment so that, for the same number of identicalamino acids, a sequence alignment with as few gaps aspossible—reflecting higher relatedness between the two comparedsequences—will achieve a higher score than one with many gaps. “Affinegap costs” are typically used that charge a relatively high cost for theexistence of a gap and a smaller penalty for each subsequent residue inthe gap. This is the most commonly used gap scoring system. High gappenalties will of course produce optimised alignments with fewer gaps.Most alignment programs allow the gap penalties to be modified. However,the default values may be used when using such software for sequencecomparisons. For example when using the GCG Wisconsin Bestfit package(see below) the default gap penalty for amino acid sequences is −12 fora gap and −4 for each extension.

Calculation of maximum % homology therefore firstly requires theproduction of an optimal alignment, taking into consideration gappenalties. A suitable computer program for carrying out such analignment is the GCG Wisconsin Bestfit package (University of Wisconsin,U.S.A.; Devereux et al., 1984, Nucleic Acids Research 12:387). Examplesof other software than can perform sequence comparisons include, but arenot limited to, the BLAST package (see Ausubel et al., 1999 ibid—Chapter18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410) and theGENEWORKS suite of comparison tools. Both BLAST and FASTA are availablefor offline and online searching (see Ausubel et al., 1999 ibid, pages7-58 to 7-60).

Although the final % homology can be measured in terms of identity, thealignment process itself is typically not based on an all-or-nothingpair comparison. Instead, a scaled similarity score matrix is generallyused that assigns scores to each pairwise comparison based on chemicalsimilarity or evolutionary distance. An example of such a matrixcommonly used is the BLOSUM62 matrix—the default matrix for the BLASTsuite of programs. GCG Wisconsin programs generally use either thepublic default values or a custom symbol comparison table if supplied(see user manual for further details). The public default values for theGCG package, or in the case of other software, the default matrix, suchas BLOSUM62 may be used.

Once the software has produced an optimal alignment, it is possible tocalculate % homology, such as % sequence identity. The softwaretypically does this as part of the sequence comparison and generates anumerical result.

Variants and Derivatives

The terms “variant” or “derivative” in relation to the amino acidsequences as described here includes any substitution of, variation of,modification of, replacement of, deletion of or addition of one (ormore) amino acids from or to the sequence. For example, the resultantamino acid sequence retains substantially the same activity as theunmodified sequence, such as having at least the same activity as themutant GnT 1 polypeptide shown in the sequence listings.

Polypeptides having the amino acid sequence shown in the Examples, orfragments or homologues thereof may be modified for use in the methodsand compositions described here. Typically, modifications are made thatmaintain the biological activity of the sequence. Amino acidsubstitutions may be made, for example from 1, 2 or 3 to 10, 20 or 30substitutions provided that the modified sequence retains the biologicalactivity of the unmodified sequence. Amino acid substitutions mayinclude the use of non-naturally occurring analogues, for example toincrease blood plasma half-life of a therapeutically administeredpolypeptide.

Natural variants of mutant GnT 1 are likely to comprise conservativeamino acid substitutions. Conservative substitutions may be defined, forexample according to the Table below. Amino acids in the same block inthe second column and in the same line in the third column may besubstituted for each other:

ALIPHATIC Non-polar GAP ILV Polar-uncharged CSTM NQ Polar-charged DE KRAROMATIC HFWY

Fragments

Polypeptides disclosed here and useful as markers also include fragmentsof the above mentioned full length polypeptides and variants thereof,including fragments of the sequences set out in the sequence listings.

Polypeptides also include fragments of the full length sequence of themutant GnT 1 polypeptide. Such fragments may comprise at least oneepitope. Methods of identifying epitopes are well known in the art.Fragments will typically comprise at least 6 amino acids, such as atleast 10, 20, 30, 50 or 100 amino acids.

Included are fragments comprising, such as consisting of, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 105, 110, 115, 120, 125, 130, 135, 140, 145 or 150, or moreresidues from a mutant GnT 1 amino acid sequence.

Polypeptide fragments of the mutant GnT 1 proteins and allelic andspecies variants thereof may contain one or more (e.g. 5, 10, 15, or 20)substitutions, deletions or insertions, including conservedsubstitutions. Where substitutions, deletion and/or insertions occur,for example in different species, such as less than 50%, 40% or 20% ofthe amino acid residues depicted in the sequence listings are altered.

Mutant GnT 1, and fragments, homologues, variants and derivatives, maybe made by recombinant means. However, they may also be made bysynthetic means using techniques well known to skilled persons such assolid phase synthesis. The proteins may also be produced as fusionproteins, for example to aid in extraction and purification. Examples offusion protein partners include glutathione-S-transferase (GST), 6×His,GAL4 (DNA binding and/or transcriptional activation domains) andβ-galactosidase. It may also be convenient to include a proteolyticcleavage site between the fusion protein partner and the proteinsequence of interest to allow removal of fusion protein sequences. Thefusion protein may be such that it does not hinder the function of theprotein of interest sequence. Proteins may also be obtained bypurification of cell extracts from animal cells.

The mutant GnT 1 polypeptide, variants, homologues, fragments andderivatives disclosed here may be in a substantially isolated form. Itwill be understood that such polypeptides may be mixed with carriers ordiluents which will not interfere with the intended purpose of theprotein and still be regarded as substantially isolated. A mutant GnT 1variant, homologue, fragment or derivative may also be in asubstantially purified form, in which case it will generally comprisethe protein in a preparation in which more than 90%, e.g. 95%, 98% or99% of the protein in the preparation is a protein.

The mutant GnT 1 polypeptides variants, homologues, fragments andderivatives disclosed here may be labelled with a revealing label. Therevealing label may be any suitable label which allows the polypeptide,etc to be detected. Suitable labels include radioisotopes, e.g. ¹²⁵I,enzymes, antibodies, polynucleotides and linkers such as biotin.Labelled polypeptides may be used in diagnostic procedures such asimmunoassays to determine the amount of a polypeptide in a sample.Polypeptides or labelled polypeptides may also be used in serological orcell-mediated immune assays for the detection of immune reactivity tosaid polypeptides in animals and humans using standard protocols.

Mutant GnT 1 polypeptide, variants, homologues, fragments andderivatives disclosed here, optionally labelled, my also be fixed to asolid phase, for example the surface of an immunoassay well or dipstick.Such labelled and/or immobilised polypeptides may be packaged into kitsin a suitable container along with suitable reagents, controls,instructions and the like. Such polypeptides and kits may be used inmethods of detection of antibodies to the polypeptides or their allelicor species variants by immunoassay.

Immunoassay methods are well known in the art and will generallycomprise: (a) providing a polypeptide comprising an epitope bindable byan antibody against said protein; (b) incubating a biological samplewith said polypeptide under conditions which allow for the formation ofan antibody-antigen complex; and (c) determining whetherantibody-antigen complex comprising said polypeptide is formed.

The mutant GnT 1 polypeptides variants, homologues, fragments andderivatives disclosed here may be used in in vitro or in vivo cellculture systems to study the role of their corresponding genes andhomologues thereof in cell function, including their function indisease. For example, truncated or modified polypeptides may beintroduced into a cell to disrupt the normal functions which occur inthe cell. The polypeptides may be introduced into the cell by in situexpression of the polypeptide from a recombinant expression vector (seebelow). The expression vector optionally carries an inducible promoterto control the expression of the polypeptide.

The use of appropriate host cells, such as insect cells or mammaliancells, is expected to provide for such post-translational modifications(e.g. myristolation, glycosylation, truncation, lapidation and tyrosine,serine or threonine phosphorylation) as may be needed to confer optimalbiological activity on recombinant expression products. Such cellculture systems in which the mutant GnT 1 polypeptide, variants,homologues, fragments and derivatives disclosed here are expressed maybe used in assay systems to identify candidate substances whichinterfere with or enhance the functions of the polypeptides in the cell.

Mutant GnT 1 Nucleic Acids

The CHO cells and cell lines comprise mutant GnT 1 nucleic acids.

We therefore provide generally for a mutant GnT 1 nucleic acid, togetherwith fragments, homologues, variants and derivatives thereof. Thesenucleic acid sequences may encode the polypeptide sequences disclosedhere, and particularly in the sequence listings.

The polynucleotide may comprise a mutant GnT 1 nucleic acid. The mutantGnT 1 nucleic acid may comprise one or more point mutations in the wildtype GnT 1 sequence. Such mutations may result in corresponding changesto the amino acid sequence, or introduce stop codons and prematuretermination of translation of the GnT 1 mRNA.

The mutant GnT 1 nucleic acid may comprise a mutation resulting in astop codon, which results in a mutant GnT 1 polypeptide being shorterthan a wild type GnT 1 polypeptide. The length of the mutant GnT 1polypeptide may be 90% or less, 80% or less, 70% or less, etc than thewild type sequence.

For example, a mutant GnT 1 nucleic acid may encode a mutant GnT 1polypeptide that is missing 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, 100 or more C-terminal residues.

The mutant GnT 1 nucleic acid may for example comprise a sequence setout in SEQ ID NO: 1. This is the mutant GnT 1 nucleic acid sequence fromthe cell line JW152. The mutant GnT 1 nucleic acid may comprise a GnT 1sequence comprising a mutation set out at column 2 of Table D1 above.

In particular, we provide for nucleic acids or polynucleotides whichencode any of the GnT 1 polypeptides disclosed here. Thus, the term “GnT1 sequence” should be construed accordingly. However, such a nucleicacid or polynucleotide may comprise a sequence set out as SEQ ID NO: 1,or a sequence encoding a of the corresponding polypeptide, and afragment, homologue, variant or derivative of such a nucleic acid. Theabove terms therefore may be taken to refer to these sequences.

As used here in this document, the terms “polynucleotide”, “nucleotide”,and nucleic acid are intended to be synonymous with each other.“Polynucleotide” generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotides” include, without limitation single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, “polynucleotide” refers to triple-stranded regions comprisingRNA or DNA or both RNA and DNA. The term polynucleotide also includesDNAs or RNAs containing one or more modified bases and DNAs or RNAs withbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications has been made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically or metabolicallymodified forms of polynucleotides as typically found in nature, as wellas the chemical forms of DNA and RNA characteristic of viruses andcells. “Polynucleotide” also embraces relatively short polynucleotides,often referred to as oligonucleotides.

It will be understood by a skilled person that numerous differentpolynucleotides and nucleic acids can encode the same polypeptide as aresult of the degeneracy of the genetic code. In addition, it is to beunderstood that skilled persons may, using routine techniques, makenucleotide substitutions that do not affect the polypeptide sequenceencoded by the polynucleotides described here to reflect the codon usageof any particular host organism in which the polypeptides are to beexpressed.

Mutant GnT 1 Variants, Derivatives and Homologues

The mutant GnT 1 polynucleotides described here may comprise DNA or RNA.They may be single-stranded or double-stranded. They may also bepolynucleotides which include within them synthetic or modifiednucleotides. A number of different types of modification tooligonucleotides are known in the art. These include methylphosphonateand phosphorothioate backbones, addition of acridine or polylysinechains at the 3′ and/or 5′ ends of the molecule. For the purposes of thepresent document, it is to be understood that the polynucleotidesdescribed herein may be modified by any method available in the art.Such modifications may be carried out in order to enhance the in vivoactivity or life span of polynucleotides.

Where the polynucleotide is double-stranded, both strands of the duplex,either individually or in combination, are encompassed by the methodsand compositions described here. Where the polynucleotide issingle-stranded, it is to be understood that the complementary sequenceof that polynucleotide is also included.

The terms “variant”, “homologue” or “derivative” in relation to anucleotide sequence include any substitution of, variation of,modification of, replacement of, deletion of or addition of one (ormore) nucleotides from or to the sequence. The resulting sequence may becapable of encoding a polypeptide which is capable of mediating highersialylation in a CHO cell.

As indicated above, with respect to sequence identity, a “homologue” hasfor example at least 5% identity, at least 10% identity, at least 15%identity, at least 20% identity, at least 25% identity, at least 30%identity, at least 35% identity, at least 40% identity, at least 45%identity, at least 50% identity, at least 55% identity, at least 60%identity, at least 65% identity, at least 70% identity, at least 75%identity, at least 80% identity, at least 85% identity, at least 90%identity, or at least 95% identity to the relevant sequence shown in thesequence listings.

There may be at least 95% identity, such as at least 96% identity, suchas at least 97% identity, such as at least 98% identity, such as atleast 99% identity. Nucleotide homology comparisons may be conducted asdescribed above. A sequence comparison program that may be used is theGCG Wisconsin Bestfit program described above. The default scoringmatrix has a match value of 10 for each identical nucleotide and −9 foreach mismatch. The default gap creation penalty is −50 and the defaultgap extension penalty is −3 for each nucleotide.

In some embodiments, a mutant GnT 1 polynucleotide has at least 90% ormore sequence identity to a sequence shown as SEQ ID NO: 1. The mutantGnT 1 polynucleotide may have 60% or more, such as 65% or more, 70% ormore, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more,97% or more or 98% or more sequence identity to a sequence shown as SEQID NO: 1.

Hybridisation

We further describe mutant GnT 1 nucleotide sequences that are capableof hybridising selectively to any of the sequences presented herein, orany variant, fragment or derivative thereof, or to the complement of anyof the above. Nucleotide sequences are such as at least 15 nucleotidesin length, such as at least 20, 30, 40 or 50 nucleotides in length.

The term “hybridisation” as used herein shall include “the process bywhich a strand of nucleic acid joins with a complementary strand throughbase pairing” as well as the process of amplification as carried out inpolymerase chain reaction technologies.

Polynucleotides capable of selectively hybridising to the nucleotidesequences presented herein, or to their complement, will be generally atleast 70%, such as at least 80 or 90% or such as at least 95% or 98%homologous to the corresponding nucleotide sequences presented hereinover a region of at least 20, such as at least 25 or 30, for instance atleast 40, 60 or 100 or more contiguous nucleotides.

The term “selectively hybridisable” means that the polynucleotide usedas a probe is used under conditions where a target polynucleotide isfound to hybridize to the probe at a level significantly abovebackground. The background hybridization may occur because of otherpolynucleotides present, for example, in the cDNA or genomic DNA librarybeing screened. In this event, background implies a level of signalgenerated by interaction between the probe and a non-specific DNA memberof the library which is less than 10•fold, such as less than 100 fold asintense as the specific interaction observed with the target DNA. Theintensity of interaction may be measured, for example, by radiolabellingthe probe, e.g. with ³²P.

Hybridisation conditions are based on the melting temperature (Tm) ofthe nucleic acid binding complex, as taught in Berger and Kimmel (1987,Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152,Academic Press, San Diego Calif.), and confer a defined “stringency” asexplained below.

Maximum stringency typically occurs at about Tm−5° C. (5° C. below theTm of the probe); high stringency at about 5° C. to 10° C. below Tm;intermediate stringency at about 10° C. to 20° C. below Tm; and lowstringency at about 20° C. to 25° C. below Tm. As will be understood bythose of skill in the art, a maximum stringency hybridisation can beused to identify or detect identical polynucleotide sequences while anintermediate (or low) stringency hybridisation can be used to identifyor detect similar or related polynucleotide sequences.

In a one aspect, we disclose nucleotide sequences that can hybridise toa mutant GnT 1 nucleic acid, or a fragment, homologue, variant orderivative thereof, under stringent conditions (e.g. 65° C. and 0.1×SSC{1×SSC=0.15 M NaCl, 0.015 M Na₃ Citrate pH 7.0}).

Where a polynucleotide is double-stranded, both strands of the duplex,either individually or in combination, are encompassed by the presentdisclosure. Where the polynucleotide is single-stranded, it is to beunderstood that the complementary sequence of that polynucleotide isalso disclosed and encompassed.

Polynucleotides which are not 100% homologous to the sequences disclosedhere but fall within the disclosure can be obtained in a number of ways.Other variants of the sequences described herein may be obtained forexample by probing DNA libraries made from a range of individuals, forexample individuals from different populations. In addition, otherviral/bacterial, or cellular homologues particularly cellular homologuesfound in mammalian cells (e.g. rat, mouse, bovine and primate cells),may be obtained and such homologues and fragments thereof in generalwill be capable of selectively hybridising to the sequences shown in thesequence listing herein. Such sequences may be obtained by probing cDNAlibraries made from or genomic DNA libraries from other animal species,and probing such libraries with probes comprising all or part of SEQ IDNO: 1 under conditions of medium to high stringency. Similarconsiderations apply to obtaining species homologues and allelicvariants of mutant GnT 1.

The polynucleotides described here may be used to produce a primer, e.g.a PCR primer, a primer for an alternative amplification reaction, aprobe e.g. labelled with a revealing label by conventional means usingradioactive or non-radioactive labels, or the polynucleotides may becloned into vectors. Such primers, probes and other fragments will be atleast 15, such as at least 20, for example at least 25, 30 or 40nucleotides in length, and are also encompassed by the termpolynucleotides as used herein. Fragments may be less than 500, 200,100, 50 or 20 nucleotides in length.

Polynucleotides such as a DNA polynucleotides and probes may be producedrecombinantly, synthetically, or by any means available to those ofskill in the art. They may also be cloned by standard techniques.

In general, primers will be produced by synthetic means, involving astep wise manufacture of the desired nucleic acid sequence onenucleotide at a time. Techniques for accomplishing this using automatedtechniques are readily available in the art.

Longer polynucleotides will generally be produced using recombinantmeans, for example using PCR (polymerase chain reaction) cloningtechniques. This will involve making a pair of primers (e.g. of about 15to 30 nucleotides) flanking a region of the sequence which it is desiredto clone, bringing the primers into contact with mRNA or cDNA obtainedfrom an animal or human cell, performing a polymerase chain reactionunder conditions which bring about amplification of the desired region,isolating the amplified fragment (e.g. by purifying the reaction mixtureon an agarose gel) and recovering the amplified DNA. The primers may bedesigned to contain suitable restriction enzyme recognition sites sothat the amplified DNA can be cloned into a suitable cloning vector.

EXAMPLES Example 1 Cell Culture

Chinese hamster ovary-K1 (CHO-K1) cells are originally obtained from Dr.Donald K. MacCallum (University of Michigan Medical Scholl, Ann Arbor,Mich.).

Parental and mutant CHO cells including JW152 cells are cultured inDulbecco's Modified Eagle Media (DMEM) supplemented with 10% fetalbovine serum (FBS) at 37° C. in a humidified incubator with 5% CO₂.

Lec1.3 cells (Lec 1 cells) are kindly provided by Dr. P. Stanley (AlbertEinstein College of Medicine, NY) and cultured in α-MEM (Gibco)supplemented with Proline (40 mg/L) (Invitrogen/Gibco) and 10% FBS.

Example 2 Isolation of RCA-I-Resistant CHO Cells

CHO-K1 cells are cultured in 6-well plates to confluence before culturemedia is changed to serum-free DMEM. Ricinus communis agglutinin I(RCA-I, EY Laboratories) is added into the media to reach a finalconcentration of 10 μg/ml and incubated with cells overnight.

Then the serum-free DMEM containing RCA-I is replaced with fresh DMEMwith 10% FBS. Nine days later, colonies of the CHO cells that survivedthe RCA-I treatment are picked and cultured in 24 well plates.

These cells are then subjected to agglutination tests using RCA-I toconfirm the mutant cells no longer react with RCA-I.

Example 3 Expression Constructs

The coding regions for human erythropoietin (EPO) and severalglycosylation related genes are cloned into the pcDNA3.1 vector(Invitrogen).

A DNA fragment that contained the open reading frame of human EPOfollowed by the internal ribosome entry site (IRES) of theEncephalomyocarditis Virus (EMCV) and the coding region for Chinesehamster N-acetylglucosaminyltransferase I (EPO-IRES-GnT I, or EIG inshort) is also cloned into pcDNA3.1. The resulting vector that expressesboth EPO and GnT I is called pEIG. To ensure efficient translation, aKozak consensus sequence (GCCACC) is placed upstream of the translationstart codon ATG in each construct.

Example 4 Transient Expression of Recombinant Human EPO in Parental andMutant CHO Cell Lines

Unless specified, 1 μg of DNA construct that expresses EPO and 1 μg ofDNA construct that expresses GnT I or another glycosylation relatedgenes are co-transfected into the wild type or JW-152 mutant cells withLipofectamine (Invitrogen) according to the manufacturer's protocols.

Two days after transfection, conditioned culture media from thetransfected cells are collected. The concentrations of recombinant EPOin each transfection sample are determined by standard ELISA using EPOELISA kits (Roche).

Example 5 Isoelectric Focusing (IEF) Analysis of EPO Produced by WildType CHO Cells and JW-152 CHO Cells

The sialylation patterns of EPO in different samples are analyzed by IEFfollowed by Western blot as previously described (Schriebl et al. 2007,Electrophoresis 28:2100-7). The pH range for IEF is 3 to 10.

Example 6 Molecular Cloning and Sequencing Analysis of GnT I cDNA inRCA-I-Resistant CHO Cells

For each cell line, 1×10⁷ cells are pelleted and rinsed in PBS. TotalRNA is extracted from the pellet using the RNAqueous kit (Ambion). cDNAis then synthesized through reverse transcription using Moloney MurineLeukemia virus (MMLV) reverse transcriptase (Promega) according to themanufacturer's recommendations

The GnT I amplicon from each cell lines' cDNA is obtained throughpolymerase chain reaction (PCR) using PFX (Invitrogen). This is thencloned into pcDNA 3.1 expression vector and sequenced. A minimum of fourclones from each mutant line are sequenced. All plasmid purificationsare carried out using mini or midi-preparation kits from Promega.Constructs are sequenced using ABI Prism 3100 Genetic Analyzer (AppliedBiosystems) after cycle sequencing with Big Dye 3.1 (AppliedBiosystems).

The results are shown at Table D1 above.

Example 7 Isolation of Stably Transfected CHO Cell Lines

CHO-K1 cells are transfected with a construct that expresses EPO alonewhilst mutant JW152 cells are transfected with pEIG to express both EPOand GnT I. The transfected cells are selected with G418 (0.8 mg/ml) fortwo weeks.

Stably transfected cells from the transfected pools are cultured in 96wells using limiting dilutions. Stable cell lines derived from singletransfected cells that expressed EPO are selected using a dot blotanalysis and the sialylation patterns of the recombinant EPO expressedby each cell line are analyzed with isoelectric focusing.

Example 8 Results: Isolation and Characterization of RCA-I-ResistantMutant CHO Cell Lines

RCA-I, a plant lectin that is known specific for β-linked galactoseresidues on glycoproteins or glycolipids, is used to selectglycosylation mutants from CHO K1 cells. Most CHO cells are killed byRCA-I after an overnight incubation.

Nine to ten days later cells that survived RCA-I treatment had growninto single clones. These clones are picked and cultured in 24 wellplates. One of the clones is named JW152.

To characterize the genetic defect in this cell line, JW152 cells aretransfected with a construct to express human EPO. The recombinant EPOproduced by JW152 cells is treated with endoglycosidase H (Endo H).

The results are shown in FIG. 1. FIG. 1 shows that JW152 expressed EPOis sensitive to Endo H treatment, suggesting that the glycan structure(N-glycans) found on the EPO molecule are of the high mannose type. Itis hence suspected that gene(s) from the early part of the glycosylationpathway is defective.

Based on this observation, a complementation test is carried out. Inthis test, in addition to the EPO construct, JW152 cells areco-transfected with several genes on the N-glycosylation pathway. Thesegenes include glucosidase I, glucosidase II, mannosidase IA, IB, IC,mannosidase II, GnT I and GnT II. EPO samples produced by JW152 cells inthese co-transfection experiments are analyzed by isoelectric focusing(IEF) followed by Western blot.

The results are shown in FIG. 2. The lane on the far left (CHO WT) showsthe EPO expressed by the wild type CHO cells as a control. The laneunder CHO-JW152 shows the EPO produced by the JW152 cells, showing theincomplete glycosylation of EPO. The rest of the lanes show the EPOproduced in JW152 cells that are co-transfected with a differentglycosylation related gene as indicated.

Among the genes tested, only GnT I is able to restore the sialylationpattern of EPO produced in JW152 cells. These results suggest that JW152cells lack functional GnT I gene.

Another important observation made from this IEF/Western blot assay isthat in the presence of GnT I, JW152 cells sialylate EPO much betterthan the wild type cells (comparing with EPO produced by the wild typecells).

To confirm that this difference is due to a unique feature of JW152cells rather than the result of over expressing GnT I, three CHO celllines are analyzed by co-transfecting them with a GnT I construct. Therecombinant EPO in conditioned media are collected and analyzed by IEF.

The results are shown in FIG. 3. GnT I did not improve EPO sialylationin wild CHO cells (comparing lanes CHO-WT and CHO-WT+GnT I). The EPOsialylation patterns of CHO-K1 with and without co-expression of GnT Iappear to be the same and so, the overexpression of GnT I is notresponsible for the betterment in sialylation seen in the lane labeledCHO-K1 plus GnT1.

GnT I dramatically improved EPO sialylation in JW152 cells (LaneJW152+GnT I). In the presence of GnT I, JW152 cells sialylate EPO muchbetter than wild type CHO cells (comparing lanes CHO-WT andCHO-JW152+GnT I).

FIG. 3 shows that, in the presence of GnT I, JW152 cells sialylaterecombinant proteins much better than the wild type cells. Treatingsamples with neuraminidase, which cleaves off sialic acid result in EPObands in the acidic region being reduced to the basic region aftercleaving off sialic acid, shows that the higher sialylated forms of EPOare indeed focused in the acidic region of the gel.

Lec1 cells are previously isolated by P. Stanley and they are known tolack GnT I activity. The IEF pattern of EPO produced by Led cells (Lec1)is similar to that of EPO produced by JW152. EPO produced by Lec1 cellsthat are co-transfected with GnT I show a similar sialylation patternwith that produced by the wild type CHO cells.

This is shown in FIG. 4. Thus, FIG. 4 shows that EPO expression in thepresence of functional GnT I of previously published Lec 1 mutant whichhas a GnT I defect is also highly sialylated.

Example 9 Results: Stable Expression of Recombinant EPO in JW152 Cellsin the Presence of GnT I

Data presented in FIG. 2 and FIG. 3 are all from transiently transfectedcells. To reveal the sialylation pattern of EPO produced by stablytransfected cells, JW152 cells are transfected with pEIG and selectedwith G418.

After two weeks of selection, single clones are picked and cultured in24 well plates for two more weeks under the selection pressure of G418.EPO produced by 10 randomly picked such stably transfected clones areanalyzed by IEF.

As shown in FIG. 10, all the EPO samples produced by different clonesare highly sialylated. The sialylation patterns are very similar to thatof EPO produced by transiently transfected cells shown in FIG. 3.

The results shown in FIG. 11 demonstrate that all the EPO samplesproduced by different stable clones are vary in overall sialylation butin general are not as well sialylated as those in FIG. 10.

Example 10 Results: All the CHO Cells that Survived RCA-I Treatment haveDysfunctional GnT I Gene and they all Sialylate their Proteins Betterwhen Co-transfected with GnT I

RCA-I is known to specifically bind β-Gal residues. Theoretically, aslong as the cells do not express β-Gal as the terminal sugar on theirsurface glycoproteins, they should survive the RCA-I treatment and beisolated as a mutant clones.

We isolated more than 100 mutant CHO clones using RCA-I and hoped thatthey should carry genetic mutations in different genes. Surprisingly,when the complementation test described in FIG. 2 is carried out, allthe clones that survived RCA-I treatment are confirmed to havedysfunctional GnT I genes.

Furthermore, as shown in FIG. 9, EPO produced in these clones are allhighly sialylated when co-transfected with GnT I. The mRNA is isolatedfrom some of the mutant lines and reverse transcribed to cDNA. Thecoding region for GnT I is amplified by PCR using the cDNA as templateand cloned into pcDNA3.1.

Sequencing analyses revealed 9 different point mutations in the GnT Igene in these mutant lines, suggesting that they arrived from differentoriginal mutant cells (see Table D1 above). Some of them, like JW152,carry a point mutation that results in a premature stop codon. Otherscarry a mutation that changes an amino acid residue in the GnT I codingregion. Nine mutant lines each with a different mutation in the GnT Igene are transfected with pEIG. EPO produced by all nine lines are allhighly sialylated when analyzed by IEF (data not shown).

Example 10 Sialylation Patterns in JW152 and Other CHO GnT1 Mutants

As shown in FIG. 5, incomplete sialylation of EPO is observed in nineCHO glycosylation mutants selected with RCA lectin, each bearing adifferent mutation in the GnT1 gene leading to a loss in GnT1 function,as compared to CHO wild-type.

As shown in FIG. 6, EPO produced in nine CHO glycosylation mutants, eachbearing a different mutation in the GnT1 gene, have a sialylationpattern that is superior to CHO wild-type after rescue.

FIG. 7 shows the results of an experiment with isoelectric focusedsamples of a different glycoprotein molecule, EPO-Fc, essentially aerythropoietin fusion protein. The experiment was conducted with aprotocol as set out above for EPO expression and IEF analysis, only withan expression vector with a different coding sequence.

The left hand lane shows expression of EPO-Fc in CHO-K1 cellstransfected with an expression vector containing EPO-Fc coding sequence.The middle lane shows expression of EPO-Fc in JW152 cells transfectedwith an expression vector containing EPO-Fc coding sequence. The righthand lane shows expression of EPO-Fc in JW152 cells co-transfected withan expression vector containing EPO-Fc coding sequence and an expressionvector containing functional GnT1 coding sequence. As shown in FIG. 7,superior sialylation by rescued JW152 is maintained even with adifferent model glycoprotein.

The results demonstrate that the superior sialylation by rescued JW152is maintained even with a different model glycoprotein.

FIG. 12 shows that the sialylation pattern observed for stable JW152cell lines in attached culture is maintained in suspension culture.

In conclusion, we have developed a method to isolate novel CHOglycosylation mutant cells. As long as RCA-I is used to treat CHO cells,the surviving cells will have a genetic defect in their GnT I gene. Whenco-transfected with GnT I, these RCA-I-resistant CHO cells expressrecombinant glycoproteins with highly sialylated N-glycans.

Example 11 HPAEC Chromatogram

FIG. 8 is a photograph showing that HPAEC chromatogram showing bettersialylated glycans cleaved from EPO-Fc expressed in JW152 cellsco-expressing functional GnT I. EPO-Fc transiently expressed in CHO-K1and JW152, contransfected with GnT I, is purified using affinitypurification via a protein A-bound chromatography column. Glycans on theequal amounts of EPO-Fc are cleaved by treatment with Peptide:N-Glycosidase F (PNGase F), and separated, using High pH Anion ExchangeChromatography (HPAEC), according to their number of sialic acidsattached to the glycans.

The chromatogram shows distinctly higher peaks for the JW152 sample inthe 4S group and a lower peak in the 1S group when comparing with theCHO-K1 sample.

REFERENCES

Weikert et al. (1999) Engineering Chinese hamster ovary cells tomaximize sialic acid content of recombinant glycoproteins. NatureBiotechnology 17:1116.

Each of the applications and patents mentioned in this document, andeach document cited or referenced in each of the above applications andpatents, including during the prosecution of each of the applicationsand patents (“application cited documents”) and any manufacturer'sinstructions or catalogues for any products cited or mentioned in eachof the applications and patents and in any of the application citeddocuments, are hereby incorporated herein by reference. Furthermore, alldocuments cited in this text, and all documents cited or referenced indocuments cited in this text, and any manufacturer's instructions orcatalogues for any products cited or mentioned in this text, are herebyincorporated herein by reference.

Various modifications and variations of the described methods and systemof the invention will be apparent to those skilled in the art withoutdeparting from the scope and spirit of the invention. Although theinvention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the claims.

The invention claimed is:
 1. A method of expressing a protein ofinterest from a Chinese Hamster Ovary (CHO) cell, the method comprising:(a) introducing a nucleic acid encoding a protein of interest into amutant Chinese Hamster Ovary (CHO) cell, wherein said mutant CHO cellcomprises: (i) a loss of function mutation in theN-Acetyl-glucosaminyltransferase I (GnT 1) gene, and (ii) an exogenousnucleic acid sequence encoding functional GnT I; and (b) allowing theprotein of interest to be expressed from the mutant CHO cell or adescendant thereof; and (c) purifying the protein.
 2. The methodaccording to claim 1, in which the protein of interest has highersialylation than a protein of interest expressed by a wild-type ChineseHamster Ovary cell.
 3. The method according to claim 1, in which theprotein of interest has a pKa of 4 or less or a Z-number of greater than150, or both.
 4. The method according to claim 1, in which the proteinof interest comprises a glycoprotein, erythropoietin (EPO), interferon-γ(IFN-γ) or Factor VIII.
 5. The method according to claim 4, in which thenucleic acid sequence encoding the protein of interest and the exogenousnucleic acid sequence encoding functional GnT I are comprised in oneexpression vector.
 6. The method according to claim 1, in which (i) themutation in the GnT I gene is selected from the following mutations atthe specified position of a GnT I sequence of GenBank Accession No.AF343963: (a) a C to T transition at nucleic acid sequence position1015; (b) a G to C transversion at nucleic acid sequence position 1300;(c) an A to C transversion at nucleic acid sequence position 638; (d) aC to G transversion at nucleic acid sequence position 784; (e) a T to Atransversion at nucleic acid sequence position 811; (f) an insertion atnucleic acid sequence position 706 resulting in a frame shift fromposition 236 of the encoded amino acid sequence; (g) a G to A transitionat nucleic acid sequence position 246; (h) a G to A transition atnucleic acid sequence position 258; or (i) an A to T transversion atnucleic acid sequence position 859, or (ii) the Chinese Hamster Ovary(CHO) cell expresses a GnT I protein comprising one or more of thefollowing mutations at the specified position of a GnT I sequence ofGenBank Accession No. AF343963: (a) Ala to Pro at amino acid position434; (b) Asp→Ala at amino acid position 213; (c) Arg→Gly at amino acidposition 262; (d) Trp→Arg at amino acid position 271; (e) frame shiftfrom position 236 resulting from an insertion at amino acid position 706of an encoding GnT I nucleic acid sequence; (f) Gln→STOP at amino acidposition 339; (g) Trp→STOP at amino acid position 82; (h) Trp→STOP atamino acid position 86; or (i) Lys→STOP at amino acid position
 287. 7.The method according to claim 1, in which the mutant Chinese HamsterOvary (CHO) cell comprises a GnT I nucleic acid sequence shown in SEQ IDNO: 1 or expresses a GnT I protein as shown in SEQ ID NO:
 2. 8. Themethod according to claim 1, in which the mutant CHO cell is comprisedin the cell line JW152 (deposited at ATCC under the Budapest Treaty asaccession number PTA-9657.
 9. The method according to claim 1, in whichthe mutant Chinese Hamster Ovary (CHO) cell comprising a mutation in theGnT 1 gene is obtained by selection with Ricinus communis agglutinin I(RCA-I).
 10. The method according to claim 9, in which the selectioncomprises culturing CHO cells in the presence of Ricinus communisagglutinin I (RCA-I) and selecting cells which survive the culture, orselecting cells which do not react with RCA-I in an agglutination test.11. The method according to claim 5, wherein said expression vector isstably transfected into the mutant CHO cell.
 12. The method according toclaim 8, wherein said cell line been adapted to suspension culture orgrowth in semi-solid medium.